Abstract
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
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Data availability
The datasets generated during and/or analyzed during the current study are not publicly available due to confidentiality and contractual obligations toward industrial partners of the project in which the data were generated but are available from the corresponding author upon reasonable request.
Code availability
The DropMap MATLAB script is available from the GitHub repository, https://github.com/LCMD-ESPCI/dropmap-analyzer. MATLAB scripts illustrating the main functions performed by the DropCell.exe MATLAB application are available from GitHub repository https://github.com/bioaster/dropcell.git. The installation file for the DropCell.exe MATLAB application, as well as an image dataset that supports/illustrates the findings of this study, are available in the figshare repository, with the identifiers https://figshare.com/articles/DropCell_exe_installer/11336663/1 and https://figshare.com/articles/dropcell_image_data_set/11342426/1, respectively.
Change history
13 January 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41596-021-00492-7
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Acknowledgements
We acknowledge the support of the REALISM study group: HCL: A. Boibieux, J. Davidson, L. Fayolle-Pivot, J. Gatel, C. Genin, A. Gregoire, A. Lepape, A.-C. Lukaszewicz, G. Marcotte, Marie Matray, D. Maucort-Boulch, N. Panel, T. Rimmele, H. Vallin; bioMérieux: S. Blein, K. Brengel-Pesce, E. Cerrato, V. Cheynet, E. Gallet-Gorius, A. Guichard, C. Jourdan, N. Koenig, F. Mallet, B. Meunier, M. Mommert, G. Oriol, C. Schrevel, O. Tabone, J. Yugueros Marcos; Bioaster: J. Becker, F. Bequet, F. Brajon, B. Canard, M. Collus, N. Garcon, I. Gorse, F. Lavocat, K. Louis, J. Moriniere, Y. Mouscaz, L. Noailles, M. Perret, F. Reynier, C. Riffaud, M.-L. Rol, N. Sapay; Sanofi: C. Carre, A. de Monfort, K. Florin, L. Fraisse, I. Fugier, M. L’Azou, S. Payrard, A. Peleraux, L. Quemeneur; ESPCI: S. Toetsch; GSK: T. Ashton, P.J. Gough, S.B. Berger, D. Gardiner, A. MacNamara, A. Raychaudhuri, R. Smylie, L. Tan, C. Tipple. This research project received funding from the French Government through the “Investissement d’Avenir” program (grant no. ANR-10-AIRT-03) and from bioMérieux. K.E. acknowledges generous funding from the ‘The Branco Weiss Fellowship – Society in Science’ and received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 80336). This work also received support from “Institut Pierre-Gilles de Gennes” (laboratoire d’excellence, “Investissements d’Avenir” programs ANR-10-IDEX-0001-02 PSL, ANR-10- EQPX-34 and ANR-10-LABX-31). This work was also supported by BPIFrance under the framework “Programme d’Investissements d’Avenir” (CELLIGO Project). The authors thank the healthy donors and the septic patients who volunteered to donate peripheral blood for these experiments. We also thank M.-N. Unheheuer, H. Laude and B.L. Perlaza for access to the BioResources platform (ICAReB). We thank F. Pène, who collected clinical samples from septic shock patients at the medical intensive care unit of Cochin Hospital (CPP17-053a / 2017-A01134-49). We thank F. Porcheray for critical reading of the manuscript. We further acknowledge the help of P. Canales Herrerias and P. Bruhns for their helpful discussion and supervision of the immunization of mice.
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Y.B., K.E., M.R. and N.A. performed and optimized the experiments described in this protocol, S.D. and G.C. provided the respective MATLAB scripts for data analysis; C.C and T.T. provided the statistical tools. M.M. optimized the boronic acid nanobead functionalization protocol. C.V. and J. Baudry managed the optical bench setup. J.-F.L. supplied the septic clinical samples. K.E., J. Bibette, J. Baudry and A.D.G. developed the DropMap technology38 and contributed to the early-stage definition of the new technology. Y.B. and C.V. extended the DropMap technology to measure low cytokine secretion profiles and to overcome limitations of cell endocytic activity. G.M., A.P. and J.T. designed and set up the clinical study involving sepsis and matched-control patients. A.T., C.G., P.L., V.M., F.V., P.C. and I.A.G. supervised the work, participated to the design of technical experiments and of the clinical study, and actively contributed in writing different sections of the manuscript. Y.B., S.D. and C.V. analyzed the data for the sepsis application, and Y.B., K.E., J. Baudry, A.D.G. and C.V. wrote the manuscript. All authors edited and proofread the paper.
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Some of the authors (J. Baudry, J. Bibette, A.D.G., Y.B. & C.V.) are inventors on patent applications based on certain ideas described in this paper and may receive financial compensation via their employers’ rewards-to-inventors schemes.
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Key references using this protocol
Eyer, K., et al. Nat. Biotechnol. 35, 977–982 (2017): https://www.nature.com/articles/nbt.3964
Rybczynska, M., et al. 38, 5337–5342 (2020): https://doi.org/10.1016/j.vaccine.2020.05.066
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Supplementary Figs. 1–3 and Supplementary Method 1 (Pipeline analysis of droplet detection and tracking).
Supplementary Data 1
Complete chip design (CAD file).
Supplementary Data 2
Mask for the double-sided tape to prepare the observation chamber.
Supplementary Data 3
Mask for laser ablation.
Supplementary Data 4
Mask for the magnet holder.
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Bounab, Y., Eyer, K., Dixneuf, S. et al. Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap. Nat Protoc 15, 2920–2955 (2020). https://doi.org/10.1038/s41596-020-0354-0
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DOI: https://doi.org/10.1038/s41596-020-0354-0
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